Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay

Abstract

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Recombinase Polymerase Amplification Based Multiplex Lateral Flow Dipstick for Fast Identification of Duck Ingredient in Adulterated Beef

“…In addition, when amplification products were carried out with MLFD test, the visualized results for different amplification products were consistent with expectations ( Figure 3 ). Compared to PCR-based methods, the whole procedure of RPA-MLFD for meat specie identification is more rapid (<35 min), easy to operate as it could be carried out at a low temperature (37–42 °C) without professional equipment, and RPA results can be directly visualized on the dipsticks [ 28 , 31 , 32 , 33 ]. Moreover, the design of RPA amplification is relatively simple, only one pair of primers and one probe are needed to complete amplification, which is more convenient than isothermal LAMP [ 34 , 35 ].…”

Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR

“…Rehbein successfully established a PCR method for detecting fish-derived DNA based on nuclear genomic DNA [29]. Wu et al established a multiplex quantitative PCR method targeting nuclear genomic DNA for the detection of fox, dog and rabbit [30]. Satkoski et al have developed a species determination assay for common poultry species based on nuclear DNA [31].…”

Establishment and application of a 10‐plex liquid bead array for the simultaneous rapid detection of animal species

“…In addition, a 15‐plex xMAP was reported to identify mammal (horse, donkey, camel, sheep, swine, red deer, buffalo, yak, dog, rat, mouse, cattle, rabbit, cat and raccoon dog), ruminants (camel, sheep, elk, buffalo and yak) and other individual species including horse, donkey, camel, sheep, swine, red deer, buffalo, yak, dog, rat, mouse, chicken and Beijing duck . Compared to these two methods, the present study adds mink and fox, which are raised for the production of fur with unrestricted doses of drugs and are reported to be substituted by some dishonest traders . Additionally, this method produced good results in terms of discriminating plant protein powder from those target meat categories that have been meat substitutes for decades.…”